Analogs of ShK Toxin and Their Uses in Selective Inhibition of Kv1.3 Potassium Channels

ABSTRACT

Analogs of ShK toxin and methods for using such ShK analogs. The ShK analogs generally comprise ShK toxin attached to a chemical entity (e.g. an atom, molecule, group, residue, compound, moiety, etc.) that has an anionic charge. In some embodiments the chemical entity attached to the ShK toxin may comprise an amino acid residue. The ShK analogs may be administered to human or non-human animal subjects to cause inhibition of potassium channels or to otherwise treat diseases or disorders. In some embodiments, the chemical entity to which the ShK toxin is attached may be chosen to provide selective inhibition of certain potassium channels (e.g., Kv1.3 channels) over other potassium channels (e.g., Kv1.1 channels). In come embodiments, the chemical entity to which the ShK toxin is attached may include a fluorophore, thereby providing a fluorophore tagged ShK analog. Such fluorophore tagged ShK analogs may be used in flow cytometry alone, or in conjunction with class II tetramers that can detect autoreactive cells.

RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 13/787,160 filed Mar. 6, 2013, which is acontinuation of U.S. patent application Ser. No. 13/286,939 filed Nov.1, 2011 and issued as U.S. Pat. No. 8,440,621, which is a continuationof U.S. patent application Ser. No. 11/663,398 filed Aug. 16, 2007 andissued as U.S. Pat. No. 8,080,523, which is a 35 U.S.C. §371 nationalstage application based on PCT International Patent Application No.PCT/US2005/036234 filed Oct. 7, 2005 which claims priority to U.S.Provosonal Patent Application No. 60/617,395 filed on Oct. 7, 2004, theentire disclosure of each such patent and patent application beingexpressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides a) novel compositions of matter, b)methods and kits for in vivo and/or in vitro inhibition of the Kv1.3channel in T- and B-lymphocytes and other cell types and c) methods fortreating autoimmune and other disorders in human or animal subjects.

BACKGROUND OF THE INVENTION

Cell plasma membranes form the outer surfaces of eukaryotic cells.Various ions (e.g., sodium, potassium, calcium, etc.) move in and out ofcells by passive diffusion through the cells' plasma membranes. Suchdiffusion of ions into and out of cells is facilitated by the presenceof “ion channels” within the cell membranes. Ion channels are proteinsembedded within the cell membrane that control the selective flux ofions across the membrane, thereby allowing for the formation ofconcentration gradients between the intracellular contents of the celland the surrounding extracellular fluid. Because ion concentrations aredirectly involved in the electrical activity of excitable cells (e.g.,neurons), the functioning (or malfunctioning) of ion channels cansubstantially control the electrical properties and behavior of suchcells. Indeed, a variety of disorders, broadly termed “channelopathies,”are believed to be linked to ion channel insufficiencies ordysfunctions.

Ion channels are referred to as “gated” if they can be opened or closed.The basic types of gated ion channels include a) ligand gated channels,b) mechanically gated channels and c) voltage gated channels. Inparticular, voltage gated channels are found in neurons, muscle cellsand non-excitable cells such as lymphocytes. They open or close inresponse to changes in the charge across the plasma membrane.

Kv1.3 Channels and Autoimmune Diseases.

Autoimmune diseases such as multiple sclerosis (MS), type-1 diabetesmellitus (T1DM), rheumatoid arthritis (RA) and psoriasis affect severalhundred million people worldwide. In these disorders specificautoreactive T cells—for instance myelin-specific T cells in MSpatients—are believed to undergo repeated autoantigen stimulation duringthe course of disease and differentiate into chronically activatedmemory cells that contribute to pathogenesis by migrating to inflamedtissues and secreting cytokines (Viglietta et al., 2002; Vissers et al.,1002; Wulff et al., 2003b). Therapies that preferentially targetchronically activated memory T cells would have significant value forautoimmune diseases.

Memory T cells are divided into two subsets—central memory (T_(CM)) andeffector memory (T_(EM))—based on the expression of the chemokinereceptor CCR7 and the phosphatase CD45RA (Geginat et al., 2001; Sallustoet al., 1999). Naïve and T_(CM) cells home to the lymph node before theymigrate to sites of inflammation, whereas T_(EM) cells home directly tosites of inflammation where they secrete copious amounts of IFN-β andTNF-α and exhibit immediate effector function. It has recently beenshown that myelin-specific autoreactive T cells in MS patients arepredominantly activated T_(EM) cells (Wulff et al., 2003b), and adoptivetransfer of myelin-specific activated rat T_(EM) cells into naïverecipients induced severe EAE (Beeton et al., 2001a; Beeton et al.,2001b). An exciting new therapeutic target for immunomodulation ofT_(EM) cells is the voltage-gated Kv1.3 K⁺ channel. T_(EM) cellsup-regulate Kv1.3 channels upon activation and their antigen-drivenproliferation is exquisitely sensitive to Kv1.3 blockers (Wulff et al.,2003b). Naïve and T_(CM) cells in contrast are significantly lesssensitive to Kv1.3 blockers to begin with and rapidly become resistantto Kv1.3 blockade by up-regulating the calcium-activated K⁺ channelIKCa1 (Ghanshani et al., 2000; Wulff et al., 2003b).

The dominance of Kv1.3 in T_(EM) cells provides a powerful way tomanipulate the activity of this subset with specific Kv1.3 inhibitors.The functionally restricted tissue distribution of the channel and thefact that in vivo Kv1.3 blockade ameliorates T_(EM)-mediated EAE, boneresorption in peridontal disease and delayed type hypersensitivityreactions in animal models without causing obvious side effects hasenhanced the attractiveness of Kv1.3 as a therapeutic target (Beeton etal., 2001b, Koo et al., 1997; Valverde et al., 2004). Although Kv1.3blockers would suppress all activated T_(EM) cells (for example T_(EM)cells specific for vaccine antigens), a Kv1.3-based therapy would be asignificant improvement over current therapies that broadly andindiscriminately modulate the entire immune system. An additionaladvantage of Kv1.3 blockers is that they are reversible. Thus, one couldtitrate the therapeutic effect of Kv1.3 blockers when needed and stoptherapy in the face of infection, unlike chemotherapeutic agents, whichtake months to subside.

Kv1.3 Channels and Obesity

The Kv1.3 channel was found to play a role in energy homeostasis andenergy balance (Hum Mol Genet. 2003 12:551-9). Mice with the Kv1.3channel genetically knocked out were able to eat fatty diets withoutgaining weight, while control mice given the same diet becameover-weight. Pharmacological blockade of Kv1.3 channels recapitulatedthe effect of genetic knockout of Kv1.3 channels. Consequently, Kv1.3blockers are likely to have use in the management of obesity.

Kv1.3 Channels and Type-2 Diabetes Mellitus.

Kv1.3 channels play a role in regulating insulin-sensitivity inperipheral target organs such as the liver and muscle (Proc Natl AcadSci USA. 2004 101:3112-7). Genetic knockout of the Kv1.3 channel in miceenhanced the sensitivity of the liver and muscle to insulin.Consequently, Kv1.3 blockers may have use in the treatment of type-2diabetes mellitus by enhancing insulin's peripheral actions and therebydecreasing blood glucose levels.

Naturally Occurring Polypeptides Known to Inhibit Kv1.3 Channels

The most potent Kv1.3 inhibitor is the peptide ShK from the Caribbeansea anemone Stichodactyla helianthus. ShK is a 35-residue polypeptidecross-linked by 3 disulfide bridges. ShK blocks Kv1.3 (K_(d)=11 pM) andsuppresses proliferation of T_(EM) cells at picomolar concentrations,and ameliorates experimental autoimmune encephalomyelitis (EAE) in ratsinduced by the adoptive transfer of myelin-specific T_(EM) cells. Apotential drawback of ShK is its low picomolar affinity for the neuronalKv1.1 channel (K_(d) 28 pM). Although no side effects were observed withShK in EAE trials, ingress of high concentrations of ShK into the brain,as might happen when the blood-brain-barrier is compromised in MS, couldlead to unwanted neurotoxicity. The development of highly specific Kv1.3inhibitors is therefore necessary. An extensive effort by thepharmaceutical industry and academic groups has yielded several smallmolecules that inhibit Kv1.3 in the mid-nanomolar range, but thesecompounds do not have the selectivity or potency to make them viabledrug candidates.

Several truncated peptidic analogs of ShK have previously been reported.In one of these ShK analogs, the native sequence was truncated and thenstabilized by the introduction of additional covalent links (anon-native disulfide and two lactam bridges). In others, non-nativestructural scaffolds stabilized by disulfide and/or lactam bridges weremodified to include key amino acid residues from the native toxin. TheseShK analogs exhibited varying degrees of Kv1.3 inhibitory activity andspecificity. Lanigan, M. D. et al.; Designed Peptide Analogues of thePotassium Channel Blocker ShK Toxin; Biochemistry, 25; 40(51):15528-37(December 2001).

There remains a need in the art for the development of new analogs ofShK that selectively inhibit Kv1.3 channels in lymphocytes with minimalor no inhibitory effects on Kv1.1 channels or other potassium channels.

SUMMARY OF THE INVENTION

The present invention provides novel compositions (referred to herein as“ShK analogs”) comprising ShK toxin attached (e.g., bound, linked by alinker or otherwise associated with) to an organic or inorganic chemicalentity (e.g. an atom, molecule, group, residue, compound, moiety, etc.)that has an anionic charge.

Further in accordance with the present invention, there are providedmethods for inhibiting potassium channels and/or treating diseases ordisorders in human or animal subjects by administering to the subject aneffective amount of an ShK analog of the present invention. In someembodiments, the chemical entity to which the ShK toxin is attached maybe chosen to provide selective inhibition of certain potassium channels(e.g., Kv1.3 channels) over other potassium channels (e.g., Kv1.1channels).

Still further in accordance with the present invention, ShK analogs ofthe foregoing character may include a fluorophore tag and suchfluorophore tagged ShK analogs of the present invention may be used inflow cytometry alone, or in conjunction with class II tetramers that candetect autoreactive cells.

Further aspects, elements and details of the present invention will beapparent to those of skill in the art upon reading the detaileddescription and examples set forth herebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of a number of ShK analogs of thepresent invention.

FIG. 2A shows a molecular model of ShK based on the published NMRstructure wherein the Lys²², critical for channel blockade, ishighlighted in one shade of grey. L-pTyr was attached to the α-aminogroup of Arg¹ of ShK (highlighted in a second shade of grey) through anAeea linker (right). The structures of the linker and L-pTyr weremodeled with AM1 in Hyperchem.

FIG. 2B shows the effect of ShK (top) and ShK(L5) (bottom) on Kv1.3 andKv1.1 currents in stably transfected cells.

FIG. 2C shows dose-dependent inhibition of Kv1.3 (open symbols) andKv1.1 (closed symbols) by ShK (dark) and ShK(L5) (light). K_(d)s onKv1.3=10±1 pM (ShK) and 69±5 pM (ShK(L5)); K_(d)s on Kv1.1=28±6 pM (ShK)and 7.4±0.8 nM (ShK(L5)).

FIG. 2D shows the time course of wash-in and wash-out of ShK(L5) onKv1.3 wherein cells were held at a holding potential of 80 mV anddepolarized for 200 msec to 40 mV every 30 secs.

FIG. 2E shows K_(d) values for inhibition of Kv1.3 and Kv1.1 by ShKanalogs. K_(d)s for ShK-F6CA and ShK-Dap²² based on published sources.

FIG. 3A is a graph showing staining intensities of CD45RA and CCR7 asdetermined by flow cytometry in the CD3⁺-gated population of human PBMCsstained with antibodies against CD3, CD45RA and CCR7.

FIG. 3B is a graph showing staining intensities of CD45RA and CCR7 asdetermined by flow cytometry in the CD3⁺-gated population in cells of ahuman T_(EM) line stained with antibodies against CD3, CD45RA and CCR7.

FIG. 3C is a graph showing the inhibitory effects of ShK (dark grey) andShK(L5) (light grey) of [³H] thymidine incorporation by PBMCs (opensymbols, a mixture of naïve/T_(CM) cells) and T_(EM) cells (closedsymbols) stimulated for 48 hours with anti-CD3 antibody.

FIG. 3D is a graphic showing of pre-activated human PBMCs (naïve/T_(CM)cells) that up-regulate KCa3.1 expression become resistant to ShK(L5)inhibition when reactivated with anti-CD3 antibody. These cells havepreviously been reported to become sensitive to the K_(Ca)3.1-specificinhibitor TRAM-34.

FIG. 4A is a graph showing CD45RC staining of rat splenic T cells (left)and PAS T cells (right) detected by flow cytometry.

FIG. 4B is a graphic showing of Kv1.3 currents exhibited by quiescent(top) and myelin antigen-activated (bottom) PAS T cells.

FIG. 4C provides a graphic representation of flow cytometry profiles ofShK-F6CA-staining in quiescent (top) and myelin antigen-activated(bottom) PAS T cells. Unstained cells (black lines) and cells stainedwith ShK-F6CA (area filled in light grey). Competition of ShK-F6CAstaining by unlabeled ShK(L5) is represented by the area filled in darkgrey.

FIG. 4D shows confocal images of Kv1.3 immunostaining in quiescent (top)and myelin antigen-activated (bottom) PAS T cells. Statistical analysiswas carried out using the Mann-Whitney U-test.

FIG. 4E shows dose-dependent inhibition by ShK (dark lines) and ShK(L5)(light lines) of [³H] thymidine incorporation by rat (left) naïve/T_(CM)(open symbols) and T_(EM) (closed symbols) cells activated with Con A (1μg/ml).

FIG. 4F shows dose-dependent inhibition by ShK (dark lines) and ShK(L5)(light lines) of IL2 secretion by PAS T cells 7 hours after stimulationwith MBP.

FIG. 4G is a graph showing that ShK(L5)-induced inhibition ofmyelin-antigen triggered [³H] thymidine incorporation by PAS T cells(open symbols) is reversed by the addition of 20 u/ml IL2 (closedsymbols).

FIG. 5A is a graph showing Kv1.3 blocking blocking activity of ShK(L5)as determined on Kv1.3 channels stably expressed in L929 cells.

FIG. 5B is a graph showing blood levels of ShK(L5) at various timesafter a single subcutaneous injection of 200 mg/kg of ShK(L5) in fourrats. Blood was drawn at the indicated times and serum was tested bypatch-clamp to determine the amount of ShK(L5).

FIG. 5C is a graph of the data of FIG. 5B fitted to a single exponentialdecay indicating a half-life of approximately 50 minutes.

FIG. 5D is a graph showing blood levels of ShK(L5) in five Lewis ratsreceiving single daily subcutaneous injections of 10 μg/kg/day ShK(L5)for 5 days. Blood was drawn each morning (24 hours after the previousinjection) and tested for blocking activity on Kv1.3 channels bypatch-clamp.

FIG. 5E is a graph showing serum levels of ShK(L5) in rats at varioustimes following a single dose of 10 mg/kg ShK(L5) either subcutaneously(open bars; n=4) or intravenously (closed bars; n=4). Blood was drawn atthe indicated times and serum was tested by patch-clamp to determine theamount of ShK(L5) in blood. ShK(L5) maintained a steady-state level of300 pM in the blood almost 24 hours after a single subcutaneousinjection. This concentration is sufficient to selectively inhibit thefunction of T_(EM) cells.

FIG. 5F is a graph showing the % recovery of ShK(L5) after ahalf-blocking dose of ShK(L5) was added to rat plasma or PBS containing2% rat plasma and incubated at 37° C. for varying duration. Aliquotswere taken at the indicated times and blocking activity determined onKv1.3 channels. ShK(L5) is extremely stable in plasma.

FIG. 6A is a graph showing scored prevention of EAE. PAS T cells wereactivated in vitro, washed, and injected intraperitoneally on day 0.Clinical scoring of EAE: 0=no clinical signs, 0.5=distal limps tail,1=limp tail, 2=mild paraparesis or ataxia, 3=moderate paraparesis,4=complete hind limb paralysis, 5=4+incontinence, 6=death. Rats(n=6/group) were injected subcutaneous with vehicle alone (n=6) orShK(L5) (n=6; 10 mg/kg/day) from day 0 to day 5.

FIG. 6B is a graph showing scored treatment of EAE. PAS T cells wereactivated in vitro, washed, and injected intraperitoneally on day 0.Treatment with ShK(L5) at 10 mg/kg/day was started when rats developedclinical signs of EAE and was continued for 3 days.

FIG. 6C is a graph showing ear thickness as an indicator of DTH reactionelicited against ovalbumin in rats. Animals (n=6/group) were treatedwith ShK(L5) 10 mg/kg/day for 2 days, after which ear swelling wasmeasured. Statistical analysis was carried out using the Mann-WhitneyU-test.

FIG. 7A shows the ShK(L5) structure and a graph showing inhibition ofKv1.3 channels in T_(EM) cells as a function of ShK(L5) concentration.Each data-point represents mean of three determinations.

FIG. 7B is a diagram of Kv1.3-containing signaling complex.

FIG. 7C shows co-localization of CD4, Kv1.3, Kvβ2, SAP97, ZIP andp56^(lck) at IS.

FIG. 7D shows CD4 and Kv1.3 staining in absence of visible T_(EM)-APCcontact.

FIG. 7E shows CD4 and Kv1.3 staining in GAD65-specific T_(EM) cellsexposed to MBP-loaded APCs.

FIG. 7F shows that ShK(L5) 100 nM does not prevent IS formation.

FIG. 7G shows that ShK(L5) 100 nM does not disrupt the IS.

FIG. 8A is a graphic showing of calcium signaling in GAD-specific T_(EM)cells from three T1DM patients triggered by anti-CD3+cross-linkingsecondary antibodies (arrow) in the absence (black) or presence ofShK(L5) 0.1 nM (dark grey), 1 nM (medium grey) or 100 nM (light grey).

FIG. 8B is a graph showing [³H]-thymidine incorporation by naïve/T_(CM)and T_(EM) cells (left) and naïve/T_(CM)-effectors and T_(EM)-effectorsfrom patients with T1DM and RA (right). T_(EM) cells: GAD65-activatedT_(EM) clones from three T1DM patients and anti-CD3 antibody activatedSF-T_(EM) cells from three RA patients. Naïve/T_(CM) cells: anti-CD3antibody-activated PB-naïve/T_(CM) cells from the same three RApatients.

FIG. 8C is a series of bar graphs showing Cytokine production by theT_(EM) and naïve/T_(CM) cells used in FIG. 8B.

FIG. 8D shows the phenotype of disease-relevant and disease-irrelevantautoreactive T cells in MS, T1DM and RA.

FIG. 8E is a diagram showing the manner in which ShK(L5) inhibitscalcium signaling, lymphocyte proliferation and cytokine production butnot IS formation.

FIG. 9 is a diagram representing a rat model of delayed typehypersensitivity (DTH) caused by effector memory T cells.

FIG. 10 is a diagram showing a treatment protocol for ShK(L5) in a ratmodel of delayed type hypersensitivity (DTH) caused by effector memory Tcells

FIG. 11 is a diagram representing specific suppression of effectormemory responses in vivo in rats by ShK(L5) without impairing thefunction of naive and central memory T cells or B cells.

FIG. 12A shows Kv1.3 currents (top) and channel number/cell (bottom) inGAD65-, insulin and myelin-specific T cells from patients with new onsettype-1 diabetes mellitus (T1DM), health controls and patients withmultiple sclerosis.

FIG. 12B shows Kv1.3 staining (top) and fluorescence intensities ofindividual T cells (bottom) from these patients.

FIG. 12C shows graphs of relative cell number vs. CCR7 stainingintensity. Cells expressing high levels of Kv1.3 are CCR7-negative i.e.they are T_(EM)-effectors. Cells expressing low levels of Kv1.3 areCCR&-positive i.e. they are either naïve or T_(CM) cells

FIG. 12D shows Kv1.3 number/cell in autoreactive T cells from a patienthaving T1DM and MS (left), patients having T1DM for greater than 5 yearsduration (middle) and patients having non-autoimmune type-2 DM.

FIG. 12E shows Kv1.3 numbers in CD4⁺GAD65-tetramer⁺ T cells from apatient with new-onset T1DM.

FIG. 13A shows Kv1.3 channel numbers per cell in peripheral T cellsblood and synovial fluid T cells of RA patients and synovial fluid Tcells of OA patients.

FIG. 13B shows confocal images of Kv1.3 (light grey) and Kvβ2 (darkergrey) staining in the cells shown in FIG. 13A.

FIG. 13C shows graphs of relative cell number vs. CCR7 stainingintensity.

FIG. 13D shows micrographs (top) and bar graphs of inflammatory index(bottom) of synovium from RA and OA patients stained with anti-CD3 oranti-Kv1.3 antibodies and counter-stained with hematoxylin/eosin (40×).

DETAILED DESCRIPTION

The following detailed description and the accompanying drawings areintended to describe some, but not necessarily all, examples orembodiments of the invention only. This detailed description and theaccompanying drawings do not limit the scope of the invention in anyway.

The present invention provides novel analogs of ShK, methods for makingsuch compositions and methods for using such compositions to inhibitKv1.3 channels (or other ion channels) in human or animal cells and fortreatment or prevention of diseases and disorders, such as T cellmediated autoimmune disorders. The compositions of the present inventioncomprise ShK toxin attached (e.g., bound, linked by a linker orotherwise associated with) to an organic or inorganic, anionic-chargedchemical entity (e.g. an atom, molecule, group, residue, compound,moiety, etc.). In at least some embodiments of the invention, theorganic or inorganic, anionic-charged chemical entity may be selected toincrease or optimize the affinity of the composition for inhibition ofKv1.3 channels over Kv1.1 channels. Examples of organic or inorganic,anionic-charged molecules or groups that may be linked or bound to ShKin accordance with the present invention include but are not necessarilylimited to:

-   -   amino acids;    -   polypeptides;    -   amino acid residues;    -   unnatural amino acid residues;    -   threonine;    -   threonine derivatives;    -   phospho-threonine;    -   serine;    -   serine derivatives;    -   phospho-serine;    -   glutamic acid;    -   glutamic acid derivatives;    -   gammacarboxy-glutamic acid;    -   aspartic acid;    -   aspartic acid derivatives;    -   inorganic compounds or groups;    -   organic compounds or groups;    -   succinic anhydride; and    -   phthalic anhydride.

In accordance with the present invention, some non-limiting examples ofcompositions of the present invention, wherein the anionic-chargedchemical entity comprises an amino acid residue, are shown in FIGS. 1and 2C and referred to herein by alphanumeric designations, as shown inTable 1 below:

TABLE 1 AMINO ACID RESIDUE BOUND TO DESIGNATION ShK AT POSITION 2 ShK-L1AEEAc-L-Pmp(OH₂) ShK-D1 AEEAc-D-Pmp(O₂) ShK-D2 AEEAc-D-Pmp(OH, Et)ShK-L3 AEEAc-L-Pmp(Et₂) ShK-D3 AEEAc-D-Pmp(Et₂) ShK-L4 AEEAc-L-TyrShK-L5 AEEAc-L-Tyr(PO₃ H₂) ShK-L6 AEEAc-L-Phe(p-NH₂) ShK-L7AEEAc-L-Phe(p-CO₂H)

With specific reference to FIG. 1, tyrosine or phenylalanine or theircharged non-natural derivatives were conjugated to ShK (top left)through a linker attached on its N terminus (residue Arg1 shown inshaded grey). The Lys22, required for channel blockade, is shown in adarker shade of grey. The molecular model of ShK is based on thepublished NMR structure and the structures of the linker and the newresidues were modeled. These embodiments of compositions of the presentinvention generally comprise the ShK toxin, which is a polypeptide,bound to (e.g., chemically bound, linked or otherwise associated with)at least one anionic-charged amino acid residue. In embodiments wherethe amino acid residue has a chiral center, the D and/or L enantiomer ofsuch amino acid residue may be used. The anionic-charged amino acidresidue may be an unnatural residue and may be attached or linked to anN-terminus of the ShK polypeptide. In some embodiments, theanionic-charged amino acid residue may be linked to an N terminus of ShKthrough a linker, such as an aminoethyloxyethyloxy-acetyl linker. Theseanalogs of ShK inhibit the Kv1.3 channel more specifically than ShKbecause they have reduced affinity for other potassium channels (e.g.,Kv1.1). The ShK may be isolated from natural sources as known in theart, or it may be synthesized.

Synthesis of ShK Toxin

ShK Toxin may be synthesized by any suitable method. In one such method,Fmoc-amino acids (Bachem Feinchemikalien) including Arg(Pmc), Asp(OtBu),Cys(Trt), Gln(Trt), His(Trt), Lys(Boc), Ser(tBu) and Thr(tBu) areobtained commercially and assembled to form ShK Toxin. Stepwise assemblyof the amino acids may be carried out on an Applied Biosystems 431Apeptide synthesizer at the 0.25 mmol scale starting withFmoc-Cys(Trt)-R. Residues 34 through 22 are single coupled. Thereafter,an aliquot (e.g., half) of the resin is removed to effect better mixing.The remainder of the peptide sequence is then double coupled to theremaining resin aliquot. All couplings are mediated bydicyclohexylcarbodiimide in the presence of 2 eq of1-hydroxybenzotriazole. The final two residues are also coupled viaHBTU/DIEA chemistry. These residues are Aeea(Fmoc-aminoethyloxyethyloxyacetic acid) and as the N-terminal residueFmoc-Tyr (PO4) monobenzyl ester. Following final removal of theFmoc-group, the peptide resin (2.42 g) is cleaved from the resin andsimultaneously deprotected using reagent K for 2 h at room temperature.Reagent K is known in the art and has been described in the literature.See, King, D. S., Fields, C. G. and Fields, G. B. (1990) Int. J. PeptideProtein Res. 36, 255-266. Following cleavage, the peptide is filtered toremove the spent resin beads and precipitated with ice cold diethylether. The peptide is then collected on a fine filter funnel, washedwith ice cold ether and finally extracted with 20% AcOH in H₂O. Thepeptide extract is subsequently diluted into 2 liters of H₂O, the pH isadjusted to 8.0 with NH₄OH and allowed to air oxidize at roomtemperature for 36 hours. Following oxidation of the disulfide bondswith a 2:1 ratio of reduced to oxidized glutathione, the peptidesolution is acidified to pH 2.5 and pumped onto a Rainin Dynamax C₁₈column (5.0×30 cm). The sample is eluted with a linear gradient from5-30% acetonitrile into H₂O containing 0.1% TFA. The resulting fractionsare analyzed using two analytical RP-HPLC systems: TFA and TEAP. Purefractions are pooled and lyophilized. (See, Pennington, M. W., Byrnes,M. E., Zaydenberg, I., Khaytin, I., de Chastonay, J., Krafte, D., Hill,R., Mahnir, V., Volberg, W. A., Gorczyca, W. and Kern, W. R. (1995) Int.J. Peptide Protein Res. 46, 354-358.)

Alternatively, solid-phase peptide synthesis employing a Boc-Bzlprotecting group strategy may be utilized to assemble the primarystructure as well as analogs of the peptide. The peptide could then becleaved from the solid-phase by anhydrous HF, yielding the linearpeptide ready for folding as described above for the Fmoc synthesizedpeptide. (See, Stewart, J. M. and Young J. D. (1984) Solid Phase PeptideSynthesis. 2^(nd) Edition. Pierce Chemical Company. Rockford, Ill.)

Alternatively, other synthetic methods to assemble the primary structureof ShK or analogs could include chemical ligation technology where thepeptide is prepared as a series of designed fragments with C-terminalthioester peptides. The the thioester peptide can react with anotherpeptide containing an N-terminal Cys residue to form a peptidecontaining a native peptide bond. By using this technology, one couldeffectively assemble the primary structure of ShK. (See, (4) Wilken, J.and Kent S. B. H. (1998) Chemical protein synthesis. Current Opin.Biotech. 9, 412-426.)

Alternatively, another synthetic method that may be employed to assemblethe primary structure of ShK would utilize a protected peptide fragmentconvergent approach as described in Albericio, F., Lloyd-Williams, P.,and Giralt, E. (1997) Convergent peptide synthesis; in Methods inEnzymol. Ed G. Fields, Academic Press, New York, N.Y. pp 313-335. Inthis method, linear protected fragments are assembled as fully sidechain protected fragments. These fragments can then be coupled togetherin a convergent manner to assemble the primary sequence of ShK or one ofits analogs. Assembly of the fragments could also utilize a solid-phaseresin to facilitate coupling and wash steps.

Alternatively, recombinant methods may be used wherein the cDNA codingsequence for ShK could be generated for expression in either aprokaryotic or eucaryotic expression system. Recombinant ShK analogscontaining unnatural amino acids are also possible by utilizing preloadtRNA molecules which utilize non-standard condons. The cCNA constructcan be engineered to use one of these unused codons to add thephosphotyrosine residue as well as the Aeea residue. Folding of therecombinantly produced ShK analog could then be accomplished in asimilar method to that used for the synthetic peptides. (See,Pennington, M. W., Byrnes, M. E., Zaydenberg, I., Khaytin, I., deChastonay, J., Krafte, D., Hill, R., Mahnir, V., Volberg, W. A.,Gorczyca, W. and Kern, W. R. (1995) Int. J. Peptide Protein Res. 46,354-358.)

Attaching Anionic Amino Acid Residues to ShK and Optional Modificationsto ShK

Anionic amino acid residues may be attached to the N terminus of naturalor synthetic ShK Toxin by way of a linker, such as anaminoethyloxyethyloxy-acetyl linker, or bay any other suitable means. Inthis example, the nine (9) ShK analogs shown in FIG. 1 are prepared.Initially, Fmoc-Aeea-OH is coupled to the N-terminus of synthetic ShKToxin assembled as described above. The resin is then divided into 9aliquots. Either Fmoc-Tyr(PO₄Bzl)-OH, Fmoc-d-Tyr(PO₄Bzl)-OH,Fmoc-Tyr(PO₄Me₂)—OH, Fmoc-Pmp-OH, Fmoc-d-Pmp-OH, Fmoc-Pmp(Et)-OH,Fmoc-Pmp(EO₂—OH, Fmoc-Tyr(tBu)-OH, or Fmoc-Amp(Boc)-OH is then coupledusing DIC and HOBT to one of the resin aliquots. The deblocked peptideresin is then cleaved and deprotected with reagent K (King et al., 1990)containing 5% triisopropylsilane for 2 h at RT. Met(O) is reduced byaddition of solid NH₄I to the cleavage cocktail at t-15 min. (Nicolas etal., 1995). For the peptide containing Tyr(PO₄Me₂)—OH, a cleavagecocktail containing 1 M TMSBr in TFA containing thioanisole as ascavenger for 18 hr at 4° C. was used (Tian et al., 1993). Incompleteremoval of the methyl protecting groups is common when using this methodand two of the species (Tyr(PO₄) and Tyr(PO₄Me)) are easily purified byRP-HPLC. The Tyr(PO₄Me₂) containing analog is cleaved via standardReagent K cleavage keeping both Me groups intact. In each case, thecleavage mixture is filtered and the crude peptide is precipitated intoice-cold diethyl ether. The precipitate is collected, yieldingapproximately 75 mg of peptide from 200 mg of resin. The crude productis dissolved in 20 ml of 50% aqueous AcOH and diluted into 0.75 I ofH₂O. The pH of the solution is adjusted with NH₄OH to 8.2, and it wasallowed to fold overnight with the addition of glutathione (2 mM:1 mM)(reduced:oxidized). All analogs are purified using RP-HPLC as describedpreviously (Pennington et al., 1995; Pennington et al., 1996a;Pennington et al., 1996b). Pure fractions are pooled and lyophilized.Each sample is confirmed by RP-HPLC, AAA and MALDI-TOF MS and adjustedto account for peptide content prior to bioassay.

In some embodiments of the invention, to improve the PK/PD properties ofthe ShK structure, residues which are sensitive to degradationproperties may be replaced or substituted. Thus, substitution of the Metresidue at position 21 may be carried out to impart a stabilizing effectto oxidation. Additionally, substitution of the C-terminal acid functionwith an amide will impart stability to C-terminal corboxypeptidaseenzymes. These two substitutions to the primary structure of ShKcombined with the anionic moiety at the N-terminus have been synthesizedto generate the most stable and selective Kv1.3 blocker. Nonhydrolyzablephosphate substitutions will also impart a stabilizing effect versusacid and basic hydrolysis of the phosphate as well as stability againstphosphatase enzymes. The substitutions are summarized below. Theacronyms used are defined as follows:Pmp=p-phosphonomethyl-Phenylalanine; Ppa=p-Phosphatityl-Phenylalanineand Nle=Norleucine.

Substitutions:

-   -   p-phospho-Tyr-Aeea-ShK-Nle21-Cys35-amide    -   p-Phosphono-methyl-Phenylalanine-Aeea-ShK-Nle21-Cys35amide (Pmp)    -   p-Phosphatityl-Phe-Aeea-ShK-Nle21-Cys35-amide (Ppa)    -   p-phospho-Tyr-Aeea-ShK-Nle21-Cys35-acid    -   p-Phosphono-methyl-Phenylalanine-Aeea-ShK-Nle21-Cys35acid (Pmp)    -   p-Phosphatityl-Phe-Aeea-ShK-Nle21-Cys35-acid (Ppa)

In addition to the nonhydrolyzable Pmp and Ppa, substitution ofp-Phosphono(difluoro-methyl)-Phenylalanine (Pfp) andp-Phosphono-methylketo-Phenylalanine (Pkp) are also anionicsubstitutions, providing the following:

-   -   Pfp-Aeea-Shk-Nle21 Cys35 amide    -   Pkp-Aeea-ShK-Nle21-Cys35 amide    -   Pfp-Aeea-Shk-Nle21 Cys35 acid    -   Pkp-Aeea-ShK-Nle21-Cys35 acid.

Structures of the N-terminal substitutions are set forth in Appendix B.Other structures that are within the scope of the present invention arepublished in Beeton, C. et al., Targeting Effector Memory T Cells with aSelective Peptide Inhibitor of Kv1.3 Channels for Therapy of AutoimmuneDiseases, Molecular Pharmacology, Vol. 67, No. 4, 1369-(2005), theentirety of which is expressly incorporated herein by reference and acomplete copy of which is appended hereto as Appendix C.

Therapeutic Uses of ShK Analogs of the Present Invention

The present invention provides methods for treating or preventingcertain disorders or diseases, such as T cell mediated disorders (e.g.,autoimmune disorders, graft vs. host disease, prevention of rejection oforgan transplants etc.), other inflammatory disorders, obesity and Type2 diabetes, in human or animal subjects by administering to the subjecta therapeutically effective (e.g., preventative or effective to reduceor eliminate symptoms or disease progression) amount of apharmaceutically acceptable preparation consisting or comprising an ShKanalog of the present invention (e.g., including but not limited tothose listed in Table 1 hereabove). Any suitable route of administration(e.g., oral, rectal, intravenous, intramuscular, subcutaneous,intradermal, intranasal, topical, transmucosal, transdermal, by drugdelivery implant, etc.) may be used. When used to prevent or treat a Tcell mediated disorder, the dosage(s) will be sufficient to inhibitKv1.3 channels on T cell membranes. In this regard, the ShK analogs ofthe present invention have the potential to be used to prevent or treata wide variety of a T cell mediated autoimmune disorders. The followingare some examples of some T cell mediated autoimmune diseases that maybe prevented or treated by the methods of the present invention,categorized with respect to the target organ that is principallyaffected by each such disease:

Nervous System: Gastrointestinal Tract: Multiple sclerosis Crohn'sDisease Myasthenia gravis Ulcerative colitis Autoimmune neuropathiesPrimary biliary cirrhosis such as Guillain-Barre Autoimmune hepatitisAutoimmune uveitis Bone resorption associated Blood: with periodontaldisease Autoimmune hemolytic anemia Endocrine: Pernicious anemia Type 1diabetes mellitus Autoimmune Addison's Disease Thrombocytopenia Grave'sDisease Vascular: Hashimoto's thyroiditis Temporal arteritis Autoimmuneoophoritis and Anti-phospholipid syndrome Orchitis Vasculitides such asMultiple Organs and/or Wegener's granulomatosis Musculoskeletal System:Behcet's disease Rheumatoid arthritis (RA) Skin: Osteoarthritis (OA)Psoriasis Systemic lupus erythematosus Dermatitis herpetiformisScleroderma Pemphigus vulgaris Polymyositis, dermatomyositis VitiligoSpondyloarthropathies such as ankylosing spondylitis Sjogren's syndrome

Irrespective of the particular organ(s) affected, T-lymphocytes arebelieved to contribute to the development of autoimmune diseases. Thecurrently available therapies for these diseases are largelyunsatisfactory and typically involve the use of glucocorticoids (e.g.methylprednisolone, prednisone), non-steroidal anti-inflammatory agents,gold salts, methotrexate, antimalarials, and other immunosuppressantssuch as cyclosporin and FK-506. Also, another T cell mediated disorderthat may be prevented or treated by the methods of the present inventionis graft vs. host disease and/or rejection of transplanted organs.Indeed, the outcomes of organ transplant procedures have progressivelyimproved with the development of refinements in tissue typing, surgicaltechniques, and more effective immunosuppressive treatments. However,rejection of transplanted organs remains a major problem. T-lymphocytesplay a central role in the immune response and they are responsible, inlarge measure, for the rejection of many transplanted organs. They arealso responsible for the so-called graft-versus host disease in whichtransplanted bone marrow cells recognize and destroy MHC-mismatched hosttissues. Accordingly, drugs such as cyclosporin and FK506 that suppressT-cell immunity are used to prevent transplant rejection andgraft-versus-host disease. Unfortunately, these T cell inhibiting drugsare toxic, with liver and renal toxicities limiting their use. Thus, themethods of the present invention may provide less toxic alternatives forthe treatment or prevention of graft vs. host disease or transplantrejection. Also, inhibitors of the voltage gated Kv1.3 potassium channelhave been shown to be especially effective in suppressing effectormemory T cells and, thus, the methods of present invention may beparticularly effective in preventing or treating diseases that areassociated with effector memory T cells, such as; bone resorption andperiodontal disease, psoriasis, rheumatoid arthritis, diabetes mellitusand multiple sclerosis. In addition to T cell mediated diseases, theKv1.3 channel has been determined to regulate energy homeostasis, bodyweight and peripheral insulin sensitivity. Thus, the methods of thepresent invention may be used to treat other diseases and disorders thatinvolve abnormal homeostasis, body weight and peripheral insulinsensitivity by inhibiting Kv1.3 channels on cell membranes, such otherdiseases and disorders include but are not necessarily limited to boneresorption in periodontal disease, Type 2 diabetes, metabolic syndromeand obesity.

Use of ShK Analogs of the Present Invention in Flow Cytometry

Further in accordance with the present invention there are providedmethods for diagnosing T cell mediated disorders or otherwise sorting ordistinguishing between various cell types in vitro using fluorophoretagged versions of ShK(L5) for use in flow cytometry alone, or inconjunction with class II tetramers that can detect autoreactive cells.Flow Cytometry is a flexible method for characterizing cells insuspension wherein fluorescence activated cell sorting is used to selectliving cells on the basis of characteristics measured by flow cytometry.The types of cellular features and functions that may be detected byflow cytometry include the expression of proteins outside and withincells, type of DNA content, viability and apoptosis, multiple drugresistance pump activity, enzyme activity, T-cell activation, T-cellreceptor specificity, cytokine expression, phagocytosis and oxidativeburst activity. Thus, in this method of the present invention, the aminoacid residue attached to the ShK may incorporate a fluorophore tag foruse in flow cytometry alone, or in conjunction with class II tetramersloaded with specific autoantigens that can detect autoreactive cells.Specific descriptions of the methods by which such flow cytometry may becarried out are described in Beeton, C., et al., A Novel FluorescentToxin to Detect and Investigate Kv1.3 Channel Up-Regulation inChronically Activated T Lymphocytes; J. Biol. Chem., Vol. 278, No. 11,9928-9937 (March 2003). In general, a flow cytometer uses focused laserlight to illuminate cells as they pass the laser beam in a fluid stream.Light scattered by the cells and light emitted by fluorescent dyesattached to cells of interest are analyzed by several detectors andprocessed by a computer. Cells may be distinguished and selected on thebasis of size and shape as well as by the presence of many differentmolecules inside and on the surface of the cells.

Examples of Potassium Channel Inhibiting Effects and Therapeutic Utilityof ShK Analogs of the Present Invention

ShK blocks the neuronal Kv1.1 channel and the Kv1.3 channel with roughlyequivalent potency. Neurotoxicity is therefore a concern undercircumstances that compromise the blood-brain-barrier and allow theentry of sufficient amounts of ShK to block Kv1.1 channels. Our strategyto design a Kv1.3-specific inhibitor was guided by our finding thatShK-F6CA containing fluorescein-6-carboxylate (F6CA) attached through a20 Å-long Aeea linker to the N-terminus of ShK exhibited 80-foldselectivity for Kv1.3 over Kv1.1 (Beeton et al., 2003). Since F6CA canexist as a restricted carboxylate or also as a cyclized lactone, it wasnot clear whether ShK-F6CA's Kv1.3 specificity was due to the negativecharge of F6CA, the hydrophobicity created by this large bulkyfluorescein nucleus, potential planar −p electronic stacking or acombination of all of these potential contributions. To distinguishbetween these possibilities and with the intention of developing anon-fluorescent Kv1.3-selective inhibitor, we generated a series of 12novel N-terminally-substituted ShK analogs to probe some of theseinteractions. By attaching tyrosine, phenylalanine or their derivatives(varying in charge, size and hydrophobicity) through an Aeea linker tothe N-terminus of ShK, we could probe the effects of charge andhydrophobicity to gain insight into our selectivity enhancement seenwith F6CA substitution.

Selective KV\v1.3 Inhibition Over Kv1.1 Inhibition:

In the example shown in FIGS. 2A-2D, L-phosphotyrosine (L-pTyr) anegatively charged (net charge 2) post-translationally modified aromaticamino acid, was attached via the AEEA linker to ShK-Arg¹ to generate thenovel analog ShK(L5). The ShK toxin and ShK(L5) were tested on Kv1.3 andKv1.1 channels stably expressed in L929 cells. FIG. 2B shows the effectsof ShK and ShK(L5) on Kv1.3 and Kv1.1 currents elicited by 200 msdepolarizing pulses from a holding potential of 80 mV to 40 mV. Bothpeptides reversibly blocked Kv1.3 and Kv1.1 in a dose-dependent mannerwith Hill coefficients of 1. K_(d)s were determined from thedose-response curves shown using Microcal Origin software. ShK blockedKv1.3 (K_(d)=10±1 pM) and Kv1.1 (K_(d)=28±6 pM) with roughly equivalentpotency as expected (FIG. 1C). In contrast, ShK(L5) was 100-foldselective for Kv1.3 (K_(d)=69±5 pM) over Kv1.1 (K_(d)=7.4±0.8 nM) (FIGS.1B, 1C). The time course of Kv1.3 current block by ShK(L5) and itswashout is shown in FIG. 1D. The time constant (T_(ON)) of ShK(L5)wash-in was 131±21 sec (n=7) while the time constant (T_(OFF)) forpeptide wash-out was 150±28 sec (n=4). The K_(d) (57±7 pM) calculatedfrom the K_(ON) (15×10⁶±0.5×10⁶ M⁻¹ sec⁻¹) and K_(OFF) (0.0059±0.0013sec⁻¹) values is consistent with the K_(d) (69±5 pM) determined withMicrocal Origin software.

Other ShK analogs were also tested on Kv1.3 and Kv1.1 channels. ShK(D5)containing D-phosphotyrosine (D-pTyr) was 35-fold selective for Kv1.3over Kv1.1 but was an order of magnitude less potent than ShK(L5).ShK(L6) containing L-pTyr-monomethyl showed modest (11-fold) Kv1.3specificity, while ShK analogs containing L-pTyr-dimethyl or L-Tyr werenot selective for Kv1.3 over Kv1.1. Analogs that contained phenylalanineor its derivatives (varying in bulk, p electron density and charge) weremodestly specific or not specific for Kv1.3 over Kv1.1. ShK(L5)'s100-fold specificity for Kv1.3 over Kv1.1 is greater than that ofShK-F6CA (80-fold), ShK(D5) (35-fold), ShK-Dap²² (33-fold) or any otherShK analog tested.

Applicants also assessed ShK(L5)'s specificity on a panel of 20 ionchannels and these data are summarized in the following Table 2:

Channels K_(d) of ShK(L5) [pM] Kv1.1  7,000 ± 1,000 Kv1.2 48,000 ± 7,000Kv1.3 (cloned) 69 ± 5 Kv1.3 (native) 76 ± 8 Kv1.4 137,000 ± 3,000  Kv1.5100,000 no effect Kv1.6 18,000 ± 3,000 Kv1.7 100,000 no effect Kv2.1100,000 no effect Kv3.1 100,000 no effect Kv3.2 20,000 ± 2,000 Kir2.1100,000 no effect Kv11.1 (HERG) 100,000 no effect K_(Ca)1.1 100,000 noeffect K_(Ca)2.1 100,000 no effect K_(Ca)2.3 100,000 no effect K_(Ca)3.1115,000 ± 5,000  Nav1.2 100,000 no effect Nav1.4 100,000 no effectSwelling-activated T cell 100,000 no effect Cl⁻ channel Cav1.2 100,000no effectAs may be appreciated from the data of Table 2 above, ShK(L5) blockedthe Kv1.3 channel in T cells with a K_(d) (76 pM) equivalent to itsK_(d) on the cloned channel (69 pM). It was 100-fold selective for Kv1.3over Kv1.1, 260-fold selective over Kv1.6, 280-fold selective overKv3.2, 680-fold selective over Kv1.2 and >1000-fold selective over allother channels tested. Importantly, it was 1600-fold Kv1.3-selectiveover KCa3.1, the calcium-activated K⁺ channel that regulates activationof human naïve and T_(CM) cells (Wulff et al., 2003). Native ShK wasless selective than ShK(L5). ShK was 2.8-fold selective for Kv1.3(K_(d)=10±1 pM) over Kv1.1 (K_(d) 28±6 pM), 20-fold selective over Kv1.6(200±20 pM), 500-fold selective over Kv3.2 (K_(d)=5,000±1,000 pM),and >1000-fold selective-over Kv1.2 (10±1 nM) and KCa3.1 (K_(d)=28±3nM). Margatoxin, a peptide from scorpion venom that has been touted as aspecific Kv1.3 inhibitor (Koo et al., 1997; Lin et al., 1993; Middletonet al., 2003) was also not specific. It was 5-fold selective for Kv1.3(110±12 pM) over Kv1.2 (K_(d)=520±1 pM), 9-fold selective over Kv1.1(10±1 nM) and >1000-fold selective over Kv1.6 and Kv3.2 (K_(d)>100 nM).Luteolin, a nutriceutical sold for autoimmune diseases (www.lutimax.com)on the basis of it being a Kv1.3 inhibitor (Lahey and Rajadhyaksha,2004), blocked Kv1.3 weakly (K_(d)=65±5 mM) and exhibited no selectivityover Kv1.1 (K_(d)=77±5 mM), Kv1.2 (K_(d)=63±4 mM) or Kv1.5 (K_(d)=41±3mM). ShK(L5)'s exquisite specificity for Kv1.3 together with itspicomolar affinity for the channel makes it a potentially attractiveimmunosuppressant.

Preferential Suppression of Human T_(EM) Cell Proliferation

With reference to FIGS. 3A-3D, in order to assess ShK(L5)'s in vitroimmunosuppressive activity, Applicants compared its ability to suppressanti-CD3 antibody-stimulated proliferation of human T_(EM) cell linesversus human PBMCs that contain a mixture of naïve and T_(CM) cells.Flow cytometry confirmed the cell surface phenotypes of the twopopulations studied. As seen in FIG. 3A, the T_(EM) lines were >90%CCR7⁻CD45RA, while as shown in FIG. 3B the PBMCs contained 65%CCR7⁺CD45RA⁺ (naïve) and 18% CCR7⁺CD45RA⁻ (T_(CM)) cells. FIG. 3C showsthat ShK(L5) and ShK were 60-fold more effective in suppressing theproliferation of T_(EM) cells (IC₅₀=˜80 pM) compared with PBMCs (IC₅₀=5nM, p<0.05). The lower sensitivity of PBMCs might be explained by arapid up-regulation of KCa3.1 channels in naïve and T_(CM) cells uponstimulation as has been reported previously (Ghanshani et al., 2000;Wulff et al., 2003). In keeping with this interpretation, PBMCsactivated for 48 hours to up-regulate KCa3.1 expression, then rested for12 hours, and re-activated with anti-CD3 antibody were completelyresistant to ShK(L5) block, as shown in the upper row of FIG. 3D. PBMCsthat had been suppressed by ShK(L5) during the first round ofstimulation exhibited identical resistance to ShK(L5) when the cellswere washed, rested and re-challenged with anti-CD3 antibody. Theseresults corroborate earlier studies indicating that naïve and T_(CM)cells escape Kv1.3 inhibitors by up-regulating KCa3.1 channels. Thus,ShK(L5) preferentially and persistently suppresses the proliferation ofT_(EM) cells.

Preferential Suppression of Rat T_(EM) Cells Proliferation

As a preamble to evaluating ShK(L5)'s therapeutic effectiveness weexamined its ability to suppress proliferation of a memory T cell line,PAS, that causes an MS-like disease in rats. As a control, Applicantsused rat splenic T cells. To confirm the differentiation status of thetwo cell populations we assessed the expression of CD45RC, a marker ofnaïve T cells (Bunce and Bell, 1997). Rat splenic T cells were 76%CD45RC⁺ (i.e. mainly naïve cells) whereas PAS cells were CD45RC⁻suggesting that they are memory cells, as shown in FIG. 4A. To determinewhether PAS cells are in the T_(EM)- or the T_(CM)-state we examinedKv1.3 expression before and 48 hours after activation. T_(EM) but notT_(CM) cells are expected to significantly up-regulate Kv1.3 levels uponstimulation. With reference to FIG. 4B, patch-clamp experiments revealeda striking increase in Kv1.3 current amplitude after MBP-stimulation ofPAS cells consistent with their being T_(EM) cells. As an independentmeasure of the number of Kv1.3 channels on PAS cells, we used ShK-F6CA,a fluorescently labeled ShK analog that. has previously been reported tobind specifically to Kv1.3. The intensity of ShK-F6CA stainingdetermined by flow cytometry reflects the number of Kv1.3 tetramersexpressed on the cell surface. As seen in FIG. 4C, ShK-F6CA (10 nM)staining intensity increased with MBP-activation of PAS cells and anexcess of unlabeled ShK(L5) (100 nM) competitively inhibited ShK-F6CAstaining. As a final test, Applicants performed confocal microscopy onquiescent and MBP-stimulated PAS cells that had been fixed and stainedwith a Kv1.3-specific antibody. In keeping with data in FIGS. 4B and 4C,resting PAS T cells had a Kv1.3 staining intensity of 4.4±0.6 and thisvalue increased to 10.6±2.3 (p<0.005) after antigen-induced activation(See FIG. 4D) showing augmentation in Kv1.3 protein expression followingactivation. Thus, MBP-activated PAS cells are CD45RC⁻ Kv1.3^(high)T_(EM) cells whereas rat splenic T cells used in our experiments arepredominantly in the naïve state.

MBP-triggered proliferation of PAS cells was suppressed ˜1000-fold moreeffectively by ShK(L5) and ShK (IC₅₀=˜80 pM) than mitogen-inducedproliferation of rat splenic T cells (See FIG. 4E, IC₅₀ ^(˜)100 nM;p<0.05). These results corroborate the findings with human T cellsdescribed above. As seen in FIG. 4G, ShK(L5) inhibited MBP-induced IL2production by PAS cells (FIG. 4F), and exogenous IL2 partially over-rodeShK(L5) suppression of PAS cell proliferation (FIG. 4G). Earlier studiesreported similar findings with less specific Kv1.3 inhibitors on human,rat and mini-pig T cells. In summary, ShK(L5) is a powerful andselective inhibitor of human and rat T_(EM) cells, and may thereforehave therapeutic use in autoimmune diseases by preferentially targetingT_(EM) cells that contribute to the pathogenesis of these disorders.

Circulating Half-Life and Stability

A patch-clamp bioassay was used to ascertain whether circulating levelsof ShK(L5) following subcutaneous injection were sufficient to inhibitT_(EM) cells. The results of these experiments are shown in FIGS. 5A-5F.

Serum samples from ShK(L5)-treated and control rats were tested forblocking activity on Kv1.3 channels. Control serum did not exhibitdetectable blocking activity indicating an absence of endogenous channelblockers. To standardize the assay, known amounts of ShK(L5) were addedto rat serum and these samples were tested on Kv1.3 channels. The spikedserum samples blocked Kv1.3 currents in a dose-dependent fashion (K_(d)77±9 pM) that was indistinguishable from ShK(L5)'s effect in the absenceof serum (FIG. 4A). Levels of ShK(L5) in treated animals were determinedby comparison with the standard curve. ShK(L5) was detectable in serum 5minutes after a single subcutaneous injection of 200 mg/kg. Peak levels(12 nM) were reached in 30 minutes and the level then fell to a baselineof about 300 pM over 420 minutes. The disappearance of ShK(L5) from theblood could be fitted by a single exponential. The circulating half-lifewas estimated to be ˜50 min.

Since the peak serum level after 200 mg/kg (12 nM) significantly exceedsthe requirement for selective blockade of Kv1.3 channels and T_(EM) cellfunction, we tested lower doses. After a single injection of 10 mg/kgthe peak serum concentration of ShK(L5) reached “500 pM within 30 min(data not shown), a concentration sufficient to block >90% Kv1.3 but notaffect Kv1.1. Repeated daily administration of this dose (10 mg/kg/day)resulted in steady-state levels of ˜300 pM (measured 24 hours afterinjection, FIG. 5D), which is sufficient to cause 60-70% suppression ofT_(EM) cells with little effect on naïve/T_(CM) cells. The“steady-state” level is unexpected given the estimated circulatinghalf-life of ˜50 min and indicates that ShK(L5) “accumulates” onrepeated administration. To determine whether the “depot” was in theskin or elsewhere in the body, we measured blood levels of ShK(L5) 10hours after rats received single intravenous or subcutaneous injectionsof 10 mg/kg ShK(L5). The peptide disappeared with the same time coursefollowing administration by either route (FIG. 5E) indicating that theskin is not responsible for the steady-state level of 300 pM ShK(L5)reached after a single 10 mg/kg daily injection (FIG. 5D), and thedepot(s) resides elsewhere.

The successful achievement of a steady-state level of 300 pM ShK(L5)following daily single injections of 10 mg/kg/day suggests that thepeptide may be stable in vivo. To directly examine its stability weincubated ShK(L5) in rat plasma or in PBS containing 2% rat plasma at37° C. for varying durations and then measured Kv1.3 blocking activity.In both sets of spiked samples (plasma and PBS) we observed a 50%reduction in Kv1.3-blocking activity in about 5 hours, presumably due topeptide binding to the plastic surface of the tube, and the level thenremained steady for the next 2-days (FIG. 5F). As an added test ofstability we compared the Kv1.3- versus Kv1.1-blocking activities ofsera from ShK(L5)-treated rats. If ShK(L5) is modified in vivo, eitherby dephosphorylation of pTyr or cleavage of the Aeea-pTyr side-chain, itwould yield ShK(L4) and ShK respectively, neither of which is selectivefor Kv1.3 over Kv1.1. Serum samples from ShK(L5)-treated animalsexhibited the same selectivity for Kv1.3 over Kv1.1 as ShK(L5),indicating that the peptide does not undergo the modifications statedabove. Taken together, these results indicate that ShK(L5) is remarkablystable in plasma and attains pharmacologically relevant serumconcentrations after single daily subcutaneous injections of 10 mg/kg.

Nontoxicity

Applicants conducted several in vitro and in vivo tests to determine ifShK(L5) exhibits any toxicity. The results of these studies aresummarized in Appendix A. Human and rat lymphoid cells incubated for 48hours with a concentration (100 nM) of ShK(L5) >1200 times greater thanthe Kv1.3 half-blocking dose or the IC₅₀ for T_(EM) suppression (70-80pM), exhibited minimal cytotoxicity. The same high concentration ofShK(L5) was negative in the Ames test on tester strain TA97A suggestingthat it is not a mutagen. Both in vitro tests failed to detect anysignificant toxicity.

Drug-induced blockade of Kv11.1 (HERG) channels has contributed to majorcardiac toxicity and the withdrawal of several medications from themarket. ShK(L5) has no effect on Kv11.1 channels at 100 nM (>1430-foldthe K_(d) for Kv1.3), and Applicants' chosen therapeutic regimen (10mg/kg/day, 300 pM steady-state circulating level) should therefore notcause cardiotoxicity. As a further test, Applicants performed heart ratevariability analysis in conscious rats administered vehicle (PBS+2% ratserum) on day-1, followed by 10 mg/kg/day ShK(L5) on day-2. ShK(L5) hadno effect on heart rate or the standard HRV (heart rate variability)parameters in both time and frequency domains (Task force of theEuropean Society of Cardiology and the North American Society of PacingElectrophysiology, 1996).

Encouraged by the acute toxicity experiments, Applicants performed asub-chronic toxicity study in which rats were administered dailysubcutaneous injections of 10 mg/kg ShK(L5) or vehicle for 2 weeks (n=6in each group). ShK(L5)-treated animals gained weight to the same degreeas rats receiving vehicle (Appendix A). Hematological and bloodchemistry analysis showed no difference between ShK(L5)- andvehicle-treated rats, and flow cytometric analysis revealed nodifferences in the proportions of thymocyte or lymphocyte subsets(Appendix A). Collectively, these studies suggest that ShK(L5) is safe.

To determine the therapeutic safety index, we administered a 60-foldhigher dose (600 mg/kg/day) of ShK(L5) to healthy rats for 5 days andobserved no clinical signs of toxicity, and no toxicity was seen whenhealthy rats received a single injection of 1000 mg/kg ShK(L5). Thesituation is less sanguine when the blood-brain-barrier is compromisedas happens in EAE and MS. Rats with EAE that received ShK(L5) 10mg/kg/day for 10 days showed no signs of toxicity. In contrast, fortypercent of rats (5/12) administered 600 mg/kg/day for five days died onthe fifth day when they developed clinical signs of EAE (extrapolatedLD₅₀=750 mg/kg/day). Since the peak concentration of ShK(L5) in theserum (12 nM) after administration of a single injection of 200 mg/kg issufficient to block >50% of Kv1.1 channels, toxicity observed in EAErats administered 600 mg/kg/day ShK(L5) is likely due to the ingressinto the brain of sufficient amounts of ShK(L5) to block Kv1.1. Thus,the effective therapeutic safety index of ShK(L5) is well in excess of100 in situations where the blood-brain barrier is not compromised (asseen in autoimmune diseases that do NOT affect the central nervoussystem (CNS)), whereas the therapeutic safety index is 75 when theblood-brain barrier is breached.

Prevention of DTH and Acute Adoptive EAE

With reference to FIGS. 6A-6C, ShK(L5) was evaluated forimmunosuppressive activity in vivo in two animal models. Applicantstested its ability to prevent and treat acute EAE induced by thetransfer of MBP-activated PAS T_(EM) cells into Lewis rats, as well asto suppress the DTH reaction mediated by T_(EM) cells. PAS cells wereactivated with MBP for 48 hours in vitro and then adoptively transferred(6-8×10⁶ viable cells) into Lewis rats. For the prevention trial, ratsthen received subcutaneous injections of saline (controls) or ShK(L5)(10 μg/kg/day) for 5 days. In the first prevention trial control ratsdeveloped mild EAE (mean maximum clinical score 2.0±1.2) with an averageonset of 5.6±0.6 days (not shown). ShK(L5) reduced disease severity(mean maximum clinical score 0.7±0.6, p<0.05). In the second preventiontrial, control rats developed more severe EAE (mean maximum clinicalscore 3.2±0.4) with a mean onset of 4.8±0.4 days (FIG. 6A). ShK(L5)significantly reduced disease severity (mean maximum clinical score0.6±0.4, p<0.007) but did not significantly delay disease onset (5.5±0.7days; p=0.07). No signs of toxicity were noted in these studies.

In the treatment trial (FIG. 6B) rats were injected with MBP-activatedPAS cells, administered saline or 10 μg/kg/day ShK(L5) when theyinitially developed signs of EAE (limp tail, hunched posture and loss of6% or more of their weight over 24 hours) and therapy was continued forthree days. Clinical signs of EAE peaked on day 6 in the control group(score=3.9±0.7) and on day 7 in the treated group (score=1.9±0.9;p<0.05).

As an independent assessment of ShK(L5)'s immunosuppressive activity invivo, Applicants also examined its effectiveness in inhibiting the DTHreaction that is mediated predominantly by skin-homing T_(EM) cells.Lewis rats immunized with ovalbumin and adjuvant were challenged 7 dayslater with ovalbumin in one ear and saline in the other ear. Rats thenreceived injections of saline (controls) or ShK(L5) (10 μg/kg/day) andear thickness was measured as an indication of DTH. All control ratsdeveloped ear swelling 24 and 48 hours after ovalbumin challenge whilethe DTH reaction was substantially milder in ShK(L5)-treated animals(FIG. 6C). Thus, ShK(L5) inhibits the T_(EM)-mediated DTH response, andprevents and ameliorates severe adoptive EAE induced by myelin-activatedT_(EM) cells.

Kv1.3 Clusters at the IS During Antigen Presentation but K⁺ EffluxThrough Kv1.3 is not Required for IS Formation or Stability

Referring to FIGS. 7A-7G, ShK(L5), a highly selective Kv1.3 inhibitor(21), blocked Kv1.3 currents in GAD65-specific T_(EM) cells with a K_(d)of 72±3 pM. We used ShK(L5) as a pharmacological probe to define thosesteps in T_(EM) cell activation that require Kv1.3 function. Biochemicalstudies have shown that Kv1.3 and Kvb2 belong to a signaling complexthat includes SAP97 (Synapse-Associated-Protein-97), ZIP(PKC-zeta-interacting-protein, p56^(lck)-associated-p62-protein, A170),p56^(lck) and CD4 (FIG. 7B). The existence of this complex in humanT_(EM) cells is supported by Applicants' results showing co-capping ofKv1.3, Kvb2, SAP97, ZIP and p56^(lck) with CD4. Furthermore, FRET(fluorescence energy transfer) studies show Kv1.3 in close proximity toCD3 in Kv1.3-transfected human T cells, and the channel preferentiallylocalizes at the point of contact between Kv1.3-transfected humancytotoxic T cells and their targets. Since CD4 traffics to the IS, thezone of contact between T cells and antigen presenting cells (APC), itis possible that Kv1.3 and other proteins in the signaling complex alsolocalize at the IS during antigen-presentation. To test this idea,GAD65-specific Kv1.3^(high) T_(EM) clones from a T1DM patient wereincubated with HLA-matched APCs that had been loaded with GAD65 557Ipeptide and stained with DAPI to aid visualization. After 20 min,APC-T_(EM) conjugates were immunostained for proteins in the signalingcomplex. CD4 co-localized at the IS with Kv1.3, Kvb2, SAP97, ZIP andp56^(lck). In the absence of APC-T_(EM) contact, CD4 and Kv1.3 weredistributed throughout the cell. Furthermore, CD4 and Kv1.3 failed tolocalize at points of contact when GAD65-specific T_(EM) cells wereexposed to APCs loaded with MBP (an irrelevant antigen), verifying thatIS-clustering is antigen-specific. Thus in GAD65-specific T_(EM) cells,a Kv1.3-containing signaling complex traffics together with CD4 to theIS during antigen-presentation, suggesting that Kv1.3 is an integralcomponent of the machinery that transduces signals in T_(EM) cells.Based on these studies, ShK(L5) at a concentration that blocksapproximately 99% of Kv1.3 channels (100 nM) did not preventIS-clustering and did not disrupt the IS once formed, indicating that K⁺efflux through Kv1.3 channels is unnecessary for IS formation orstability.

Suppression of Human T_(EM) Cells

With reference to FIGS. 8A-8E, ShK(L5) inhibited calcium signaling inT_(EM) cells, an early and essential step in T cell activation.GAD65-specific T_(EM) clones from T1DM patients were loaded with thecalcium indicator dye Fluo3, pre-incubated in medium alone or withincreasing concentrations of ShK(L5) and imaged by flow cytometry beforeand after the addition of an activating anti-CD3 antibody and across-linking secondary antibody. Peak calcium rise occurred in 242±35seconds after stimulation and was blocked by ShK(L5) with an IC₅₀ of˜200 pM (FIG. 8A). ShK(L5) was 10-fold more effective in suppressing[³H]-thymidine-incorporation by autoreactive T_(EM) cells from T1DM andRA patients compared with naïve/T_(CM) cells from these patients (FIG.8B, left). In a second set of experiments (FIG. 8B, right), RA-SF andRA-PB T cells were activated with anti-CD3 antibody for 48 hours togenerate “T_(EM)-effectors” and “naïve/T_(CM)-effectors” respectively.Cells were rested overnight in medium, re-stimulated with anti-CD3antibody in the presence or absence of ShK(L5) for a further 48 hoursand [³H]-thymidine incorporation was measured. RA-SF-T_(EM)-effectorsretained their sensitivity to ShK(L5) inhibition, whereasRA-PB-naïve/T_(CM)-effectors were resistant to Kv1.3 blockade (FIG. 8B,right), most likely because they up-regulate the calcium-activatedKCa3.1/IKCa1 channel, which substitutes for Kv1.3 in promoting calciumentry. ShK(L5) profoundly suppressed the production of interleukin 2(IL2) and interferon-g (IFN-g) by T_(EM) cells from T1DM and RApatients, whereas IL2 and IFN-g production by naïve/T_(CM) cells fromthese patients was less affected (FIG. 8C). The production of tumornecrosis factor-a and interleukin 4 by both T_(EM) cells andnaïve/T_(CM) cells was less sensitive to ShK(L5) (FIG. 8C).

Verification of Rat Model of Delayed Type Hypersensitivity (DTH) Causedby Effector Memory T Cells.

As shown in FIG. 9, rats were immunized with ovalbumin (OVA) inadjuvant. They were challenged in one ear 7 days later with OVA and inthe other ear with saline. Ear swelling was measured 24 h later as asign of delayed type hypersensitivity (DTH). The FACS histograms shownin FIG. 9 indicate that T cells in the ears challenged with OVA areCD45RC-negative memory cells while T cells in the blood and spleen ofthe same rats are mostly naive T cells.

Treatment Protocol for Shk(L5) in a Rat Model of Delayed TypeHypersensitivity (DTH) Caused by Effector Memory T Cells

As shown in FIG. 10, rats received ShK(L5) 10 μg/kg/day as asubcutaneous injection either from day 0 to day 7 (during the primingphase) to prevent the differentiation of naiv cells to effector memoryTEM cells, or during the effector phase after challenge to the ear withovalbumin to prevent the function of the TEM cells.

Shk(L5) Specifically Suppresses Effector Memory Responses in Vivo inRats without Impairing the Function of Naive and Central Memory T Cellsor B Cells

As shown in FIG. 11, control rats developed ear swelling i.e. a positiveDTH response. ShK(L5) was NOT effective in suppressing DTH whenadministered during the priming phase, indicating that it did notsuppress the differentiation of naive and central memory T cells intoeffector memory cells. ShK(L5) suppressed DTH when administered duringthe effector phase indicating that it either prevented the ability ofeffector memory T cells to reach the ear and/or suppressed theactivation of effector memory T cells. The first possibility wasexcluded because the number of T cells in the ears of ShK(L5)-treatedrats was the same as in the ears of rats given the vehicle. ShK(L5)suppressed effector memory T cell activation in the ear because these Tcells were Kv1.3-negative, while the memory T cells in the ears ofvehicle-treated animals were Kv1.3 positive. IgM and IgG B-cellresponses in these animals was also not affected.

Kv1.3 Expression in T Cells Specific for GAD65/555-567, Insulin/9-23-and Myelin Antigens from Patients with T1DM or MS and Healthy Controls

FIG. 12A shows Kv1.3 currents (top) and channel number/cell (bottom)from antigen-specific T cells from patients with new onset type-1diabetes mellitus, health controls and patients with multiple sclerosis.Each data-point represents the mean±SEM from 20-50 cells from 2-4 T celllines from a single donor measured 48 hours after the third antigenstimulation. Due to the low frequency of T cells specific for insulinand GAD65 in the blood of T1DM patients and controls, we amplified thesepopulations by generating short-term autoantigen-specific CD4⁺ T celllines using the split-well method. As controls, we generated T celllines specific for the irrelevant autoantigen myelin basic protein (MBP)that is implicated in MS but not T1DM. Following the third antigenicstimulation, Kv1.3 currents were measured by whole-cell patch-clamp inactivated cells with a membrane capacitance greater than 4 pF (celldiameter≧11 μm). Representative Kv1.3 currents and Kv1.3channel-numbers/T cell are shown in FIG. 12A. The currents displayedbiophysical and pharmacological properties characteristic of Kv1.3. Tcells specific for insulin (9-23) or GAD65 (555-567) from patients withnew onset T1DM displayed large Kv1.3 currents and expressed high numbersof Kv1.3 channels, whereas disease-irrelevant MBP-specific T cells fromthese patients were Kv1.3^(low) (p=0.001). For comparison we haveplotted our published Kv1.3 data on MS patients in whom the oppositepattern was observed. In MS patients, T cells specific for MBP or myelinoligodendrocyte glycoprotein (peptide 35-55) or proteolipid protein(peptide 139-151) were Kv1.3^(high), while insulin- and GAD65-specific Tcells were Kv1.3^(low) (p=0.0001). Autoreactive T cells isolated fromhealthy controls were Kv1.3^(low) regardless of specificity. In oneindividual with both MS and T1DM, T cells specific for all threeautoantigens were Kv1.3^(high). GAD65-specific and insulin-specific Tcells from patients with longstanding T1DM were Kv1.3^(high) reflectingthe persistence of autoreactive T_(EM) cells, whereas a Kv1.3^(low)pattern was found in GAD65- and insulin-specific T cells from patientswith non-autoimmune type-2 DM. As seen in FIG. 12B, Kv1.3 staining (top)and fluorescence intensities of individual cells (bottom). Applicantsconfirmed the patch-clamp data by immunostaining for Kv1.3. Insulin- andGAD65-specific T cells from T1DM patients and MBP-specific T cells fromMS patients stained brightly whereas cells specific for irrelevantautoantigens stained dimly. FIG. 12C shows CCR7 expression. Flowcytometry revealed that Kv1.3^(high) T cells were CCRT T_(EM) cells,while Kv1.3^(low) cells were CCR7⁺ naïve or T_(CM) cells. FIG. 12D showsKv1.3 number/cell in autoreactive T cells from a patient with both T1DMand MS, and from patients having T1DM or type-2 DM for greater than 5years and 2 years, respectively. FIG. 12E shows Kv1.3 numbers inCD4⁺GAD65-tetramer⁺ T cells from patient with new-onset T1DM. As afurther control, we used fluorescent MHC class II tetramers containingthe GAD65 557I peptide, to isolate GAD65-specific CD4⁺ T cells from aDR-0401-positive patient with new onset T1DM. Tetramer-sortedGAD65-activated T cells displayed the same Kv1.3^(high) pattern observedin GAD65-specific T cell lines from T1DM patients. In summary,disease-relevant, autoantigen-activated T cells in both T1DM and MS areKv1.3^(high) CCRT T_(EM)-effectors, while disease-irrelevantautoreactive cells in these patients are Kv1.3^(low)CCR7⁺ naïve/T_(CM)cells.

Kv1.3 Expression in Rheumatoid Arthritis and Osteoarthritis

In RA, disease-relevant T cells can be isolated from affected joints.Applicants patch-clamped T cells from the synovial fluid (SF) of 7 RApatients 48 hours after stimulation with anti-CD3 antibody. As seen inFIG. 13A, as controls Applicants analyzed SF-T cells from 7 patientswith degenerative, non-autoimmune osteoarthritis (OA) (which had beenactivated with the same protocol. RA-SF T cells were Kv1.3^(high)whereas OA-SF T cells were Kv1.3^(low) (p<0.0001). Applicants found theKv1.3^(low) pattern in anti-CD3-activated T cells from the peripheralblood (PB) of RA patients (p<0.0001) because autoreactive Kv1.3^(high)T_(EM) cells are infrequent in the blood. Immunostaining for Kv1.3 andits associated Kvβ2 subunit corroborated the patch-clamp data. FIG. 13Bshows confocal images of Kv1.3 (light grey as seen in the figure) andKvβ2 (darker grey as seen in the figure) staining. RA-SF T cells stainedbrightly for both Kv1.3 and Kvβ2, while OA-SF and RA-PB T cellsdisplayed weak staining. FIG. 13C illustrates CCR7 expression. Flowcytometry verified that Kv1.3^(high) RA-SF T cells were CCRT T_(EM)cells, while Kv1.3^(low) OA-SF and RA-PB T cells were CCR7⁺ naïve/T_(CM)cells. FIG. 13D (top) shows micrographs of synovium from RA and OApatients stained with anti-CD3 or anti-Kv1.3 antibodies andcounter-stained with hematoxylin/eosin (40×). As a further test, weimmunostained paraffin-embedded synovial tissues (ST) from 5 RA and 5 OApatients for CD3, Kv1.3 and CCR7. We have previously shown that ourstaining method does not detect Kv1.3 in naïve/T_(CM) cells because oftheir low numbers of Kv1.3 channels. In RA-ST, a preponderance ofCD3⁺Kv1.3⁺CCR7⁻ T_(EM) cells was seen, whereas CD3⁺ cells were sparse inOA-synovium and these were mainly Kv1.3⁻CCR7⁺ naïve/T_(CM) cells. Degreeof infiltration by CD3⁺, Kv1.3⁺ and CCR7⁺ cells assessed by gradingsystem in Figure S2A. CD3⁺-inflammatory-index: RA=3.2±0.1; OA=1.1±0.2(p<0.01); Kv1.3⁺-inflammatory-index: RA=2.8±0.3; OA=0.6±0.3 (p<0.01).Thus, in three different autoimmune disorders, our results areconsistent with disease-associated autoreactive T cells beingKv1.3^(high) CCR7⁻ T_(EM)-effectors.

It is to be appreciated that the invention has been described hereinwith reference to certain examples or embodiments of the invention butthat various additions, deletions, alterations and modifications may bemade to those examples and embodiments without departing from theintended spirit and scope of the invention. For example, any element orattribute of one embodiment or example may be incorporated into or usedwith another embodiment or example, unless to do so would render theembodiment or example unsuitable for its intended use. Also, where thesteps of a method or procedure are listed or stated in a particularorder, the order of those steps may be changed unless otherwisespecified or unless such change in the order of the steps would renderthe invention unpatentable or unsuitable for its intended use. Allreasonable additions, deletions, modifications and alterations are to beconsidered equivalents of the described examples and embodiments and areto be included within the scope of the following claims.

APPENDIX A Toxicity study of ShK(L5) In vitro tests 100 nM ShK(L5)Cytotoxicity (% dead cells) Human PBMCs 7.5 ± 4.3 PAS T cells 8.1 ± 0.8Jurkat cells 5.5 ± 3.3 Burkitt lymphoma 3.1 ± 0.9 RPMI 8226 myeloma 6.5± 2.1 Ames Test Negative Acute in vivo tests Saline ShK(L5) 10 μg/kgElectrocardiogram* Heart rate 302 ± 13 311 ± 20  SDNN 13.3 ± 3.0 17.8 ±4.4  CV %  6.7 ± 1.4 9.2 ± 2.2 SDANN_(5 min)  5.0 ± 2.0 6.9 ± 2.3 rMSSD 6.8 ± 2.2 9.8 ± 3.5 HF (n.u.)  71 ± 21 79 ± 37 HF (%) 50 ± 8 53 ± 10 LF(n.u.) 68 ± 4 64 ± 10 LF (%) 50 ± 8 47 ± 10 LF/HF  1.1 ± 0.4 1.3 ± 0.7ShK(L5) 10 μg/kg/day Sub-chronic in vivo tests Saline for 2 weeks Weightgain (%)  7.2 ± 1.8  6.2 ± 1.7 Complete blood count Hematocrit (%) 40.3± 1.4 39.0 ± 4.9 Hemoglobin (g/dl) 15.3 ± 0.5 15.0 ± 1.5 MCV (fl) 48.5 ±0.2 48.3 ± 0.3 MCH (pg) 18.5 ± 0.8 18.5 ± 0.6 MCHC (g/dl) 38.0 ± 1.838.4 ± 1.3 Total white cells (×10³ mm⁻³)  7.1 ± 2.1  7.1 ± 2.5 Total redcells (×10⁶ mm⁻³)  8.3 ± 0.3  8.1 ± 1.0 Total platelets (×10³ mm⁻³)  656± 214  606 ± 106 Blood chemistry Alkaline phosphatase (U/I) 170 ± 26 150± 18 Glucose (mg/dl) 139 ± 21 150 ± 18 Blood urea nitrogen (mg/dl) 17.1± 2.6 15.0 ± 1.7 Creatinine (mg/dl) 0.6 ± 0   0.6 ± 0.1 Albumin (g/dl) 5.0 ± 0.3  4.5 ± 0.4 Thymic cell populations (%) CD4⁻CD8⁻  3.6 ± 1.1 4.3 ± 0.7 CD4⁺CD8⁺ 77.8 ± 6.1 76.8 ± 4.1 CD4⁺CD8⁻  8.5 ± 1.7 11.2 ± 2.0CD4⁻CD8⁺ 10.0 ± 3.3  7.6 ± 1.3 CD3⁺ 89.5 ± 1.6 93.2 ± 3.5 Splenicpopulations (%) CD3⁺ 72.4 ± 4.4 65.4 ± 0.1 CD3⁺CD45RC⁺ 35.6 ± 2.6 39.8 ±1.1 CD3⁺CD45RC⁻ 23.6 ± 2.3 26.5 ± 1.3 CD3⁺CD4⁺ 62.7 ± 0.1 66.6 ± 1.2CD3⁺CD8⁺ 26.9 ± 0.1 25.0 ± 0.2 IgM⁺ 38.8 ± 1.5 33.3 ± 0.3 Data expressedas mean ± SD. *Tested with t-tests, p < 0.05 on all parameters; SDNN;Standard deviation of all normal-to-normal RR intervals; CV %: 100 ×SDNN/average RR interval; SDANN_(5 min): Standard deviation of the meanof normal RR intervals for each 5 min period; rMSSD: Root mean square ofsuccessive difference; HF (n.u.): High frequency (0.75-2.5 Hz) power innormalized unit; LF (n.u.): Low frequency (0.2-0.75 Hz) power innormalized unit.

1-21. (canceled)
 22. A method for causing inhibition of Kv1.3 potassiumchannels in a human or animal subject, said method comprising the stepof: (A) administering to the subject a composition that comprises ShKtoxin attached to an organic or inorganic chemical entity that has ananionic charge, in a form and amount that is effective to inhibit ofKv1.3 potassium channels.
 23. A method according to claim 22 wherein themethod is carried out to prevent or treat an autoimmune disorder.
 24. Amethod according to claim 23 wherein the autoimmune disorder is selectedfrom the group consisting of: Multiple sclerosis Myasthenia gravisAutoimmune neuropathies such as Guillain-Barré Autoimmune uveitisCrohn's Disease Ulcerative colitis Primary biliary cirrhosis Autoimmunehepatitis Autoimmune thrombocytopenia Type 1 diabetes mellitus Addison'sDisease Grave's Disease Hashimoto's thyroiditis Autoimmune oophoritisand orchitis Behcet's disease Rheumatoid arthritis bone resorptionassociated with periodontal disease Systemic lupus erythematosusScleroderma Polymyositis, dermatomyositis Pemphigus vulgarisSpondyloarthropathies such as ankylosing spondylitis Sjogren's syndrome25. A method according to claim 22 wherein the method is carried out toprevent or treat grail vs. host disease.
 26. A method according to claim22 wherein the method is carried out to treat or prevent rejection of atransplanted tissue or organ.
 27. A method according to claim 22 whereinthe method is carried out to prevent or treat metabolic syndrome.
 28. Amethod according to claim 22 wherein the method is carried out to treator prevent Type 2 diabetes.
 29. A method according to claim 22 whereinthe method is carried out to treat or prevent obesity.
 30. A methodaccording to claim 22 wherein the method is carried out to treat orprevent bone resorption associated with periodontal disease.
 31. Amethod according to claim 22 wherein the composition comprises ShK toxinattached to a chemical entity selected from the group consisting of:amino acids; polypeptides; amino acid residues; unnatural amino acidresidues; threonine; threonine derivatives; phospho-threonine; serine;serine derivatives; phospho-serine; glutamic acid; glutamic acidderivatives; gammacarboxy-glutamic acid; aspartic acid; aspartic acidderivatives; inorganic compounds or groups; organic compounds or groups;succinic anhydride; and phthalic anhydride.
 32. A method according toclaim 22 wherein the chemical entity is AEEAc-L-Pmp(OH2).
 33. A methodaccording to claim 22 wherein the chemical entity is AEEAc-D-Pmp(OH2).34. A method according to claim 22 wherein the chemical entity isAEEAc-D-Pmp(OH, Et).
 35. A method according to claim 22 wherein thechemical entity is AEEAc-L-Pmp(Et2).
 36. A method according to claim 22wherein the chemical entity is AEEAc-D-Pmp(Et2).
 37. A method accordingto claim 22 wherein the chemical entity is AEEAc-L-Tyr.
 38. A methodaccording to claim 22 wherein the chemical entity is AEEAc-L-Tyr(PO3H2).39. A method according to claim 22 wherein the chemical entity isAEEAc-L-Phe(p-NH2).
 40. A method according to claim 22 wherein thechemical entity is AEEAc-L-Phe(p-CO2H).
 41. A method according to claim22 wherein the chemical entity is AEEAc-L-Aspartate. 42.-54. (canceled)